Nuclear factor-erythroid 2-related factor 2 (Nrf2/NFE2L2), a redox-sensitive transcription factor plays a critical role in adaptation to cellular stress and affords cellular defense by initiating transcription of antioxidative and detoxification genes. While a protein can be regulated at multiple levels, control of Nrf2 has been largely studied at post-translational regulation points by Keap1. Importantly, post-transcriptional/translational based regulation of Nrf2 is less understood and to date there are no reports on such mechanisms in neuronal systems. In this context, studies involving the role of microRNAs (miRs) which are normally considered as fine tuning regulators of protein production through translation repression and/or post-transcriptional alterations, are in place. In the current study, based on in-silico analysis followed by immunoblotting and real time analysis, we have identified and validated for the first time that human NFE2L2 could be targeted by miR153/miR27a/miR142-5p/miR144 in neuronal, SH-SY5Y cells. Co-transfection studies with individual miR mimics along with either WT 3′ UTR of human Nrf2 or mutated miRNA targeting seed sequence within Nrf2 3′ UTR, demonstrated that Nrf2 is a direct regulatory target of these miRs. In addition, ectopic expression of miR153/miR27a/miR142-5p/miR144 affected Nrf2 mRNA abundance and nucleo-cytoplasmic concentration of Nrf2 in a Keap1 independent manner resulting in inefficient transactivating ability of Nrf2. Furthermore, forced expression of miRs diminished GCLC and GSR expression resulting in alteration of Nrf2 dependent redox homeostasis. Finally, bioinformatics based miRNA-disease network analysis (MDN) along with extended computational network analysis of Nrf2 associated pathologic processes suggests that if in a particular cellular scenario where any of these miR153/miR27a/miR142-5p/miR144 either individually or as a group is altered, it could affect Nrf2 thus triggering and/or determining the fate of wide range of disease outcomes.

Background Prenatal exposure to ethanol elicits a range of neuro-developmental abnormalities, microcephaly to behavioral deficits. Impaired protein synthesis has been connected to pathogenesis of ethanol-induced brain damage and abnormal neuron development. However, mechanisms underlying these impairments of protein synthesis are not known. In this study, we illustrate the effects of ethanol on programmed cell death protein 4 (PDCD4), a tumor and translation repressor. Methods Primary cortical neurons (PCNs) were treated with ethanol for 2.5mg/ml and 4mg/ml for different time points (4h to 24h) and PDCD4 expression was detected by Western blotting. Protein synthesis was determined using [35S] methionine incorporation assay. Methyl cap pull down assay was performed to establish the effect of ETOH on association of eIF4A with capped mRNA. Luciferase assay was performed to determine the in vivo translation. A 2-day acute 5-dose binge model with ethanol (4 g/kg body wt, 25% v/v) was performed in Sprague Dawley rats at 12h intervals and analyzed for PDCD4, eukaryotic initiation factor 4A (eIF4A) and eIF4A-methyl cap association. Results Ethanol increased PDCD4 expression in a time- and dose-dependent manner in PCNs which inhibited the association of eIF4A with methyl-cap. Ethanol and ectopic PDCD4 expression suppressed in vivo translation in PCNs and RNAi targeting of PDCD4 blocked the inhibitory effect of ethanol on protein synthesis. In utero exposure of pregnant rats to ethanol resulted in a significant increase of PDCD4 protein in fetal cerebral cortex along with inhibition of methyl-cap associated eIF4A, compared to iso-caloric controls. Increased PDCD4 also occurred in pooled fractions of remaining brain regions. Conclusions Our data for the first time, illustrate that PDCD4 mediates inhibitory effects of ethanol on protein synthesis in PCNs and developing brain.

Glutathione (GSH), a major cellular antioxidant protects cells against oxidative stress injury. Nuclear factor erythroid 2-related factor 2 (NFE2L2/Nrf2) is a redox sensitive master regulator of battery of antioxidant enzymes including those involved in GSH antioxidant machinery. Earlier we reported that ethanol (ETOH) elicits apoptotic death of primary cortical neurons (PCNs) which in partly due to depletion of intracellular GSH levels. Further a recent report from our laboratory illustrated that ETOH exacerbated the dysregulation of GSH and caspase mediated cell death of cortical neurons that are compromised in Nrf2 machinery (Narasimhan et al., 2011). In various experimental models of neurodegeneration, neuronal antioxidant defenses mainly GSH has been shown to be supported by astrocytes. We therefore sought to determine whether astrocytes can render protection to neurons against ETOH toxicity, particularly when the function of Nrf2 is compromised in neurons. The experimental model consisted of co-culturing primary cortical astrocytes (PCA) with Nrf2 downregulated PCNs that were exposed with 4 mg/mL ETOH for 24 h. Monochlorobimane (MCB) staining followed by FACS analysis showed that astrocytes blocked ETOH induced GSH decrement in Nrf2-silenced neurons as opposed to exaggerated GSH depletion in Nrf2 downregulated PCNs alone. Similarly, the heightened activation of caspase 3/7 observed in Nrf2-compromised neurons was attenuated when co-cultured with astrocytes as measured by luminescence based caspase Glo assay. Furthermore, annexin-V-FITC staining followed by FACS analysis revealed that Nrf2 depleted neurons showed resistance to ETOH induced neuronal apoptosis when co-cultured with astrocytes. Thus, the current study identifies ETOH induced dysregulation of GSH and associated apoptotic events observed in Nrf2-depleted neurons can be blocked by astrocytes. Further our results suggest that this neuroprotective effect of astrocyte despite dysfunctional Nrf2 system in neurons could be compensated by astrocytic GSH supply.